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Yorodumi- EMDB-4757: Subtomogram average of cytosolic ribosomes after cryo-FIB lift-ou... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-4757 | |||||||||
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Title | Subtomogram average of cytosolic ribosomes after cryo-FIB lift-out (p1 state) | |||||||||
Map data | Subtomogram average of cytosolic ribosomes after cryo-FIB lift-out (p1 state) | |||||||||
Sample |
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Biological species | Caenorhabditis elegans (invertebrata) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 11.8 Å | |||||||||
Authors | Pfeffer S / Mahamid J / Albert S / Plitzko JM | |||||||||
Funding support | Germany, 2 items
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Citation | Journal: Nat Methods / Year: 2019 Title: A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue. Authors: Miroslava Schaffer / Stefan Pfeffer / Julia Mahamid / Stephan Kleindiek / Tim Laugks / Sahradha Albert / Benjamin D Engel / Andreas Rummel / Andrew J Smith / Wolfgang Baumeister / Juergen M Plitzko / Abstract: Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows ...Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, this approach has been mainly limited to individual cells that can be completely vitrified by plunge-freezing. Here, we describe a preparation method that is based on the targeted extraction of material from high-pressure-frozen bulk specimens with a cryo-gripper tool. This lift-out technique enables cryo-electron tomography to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology. We demonstrate the potential of the lift-out technique with a structural study of cytosolic 80S ribosomes in a Caenorhabditis elegans worm. The preparation quality allowed for subtomogram analysis with sufficient resolution to distinguish individual ribosomal translocation states and revealed significant cell-to-cell variation in ribosome structure. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_4757.map.gz | 7.4 MB | EMDB map data format | |
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Header (meta data) | emd-4757-v30.xml emd-4757.xml | 10.2 KB 10.2 KB | Display Display | EMDB header |
Images | emd_4757.png | 79.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-4757 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-4757 | HTTPS FTP |
-Validation report
Summary document | emd_4757_validation.pdf.gz | 260.7 KB | Display | EMDB validaton report |
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Full document | emd_4757_full_validation.pdf.gz | 259.8 KB | Display | |
Data in XML | emd_4757_validation.xml.gz | 5.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4757 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4757 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_4757.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Subtomogram average of cytosolic ribosomes after cryo-FIB lift-out (p1 state) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.42 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Cytosolic ribosome in the native cellular environment after cryo-...
Entire | Name: Cytosolic ribosome in the native cellular environment after cryo-FIB lift-out from C. elegans (p1 state) |
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Components |
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-Supramolecule #1: Cytosolic ribosome in the native cellular environment after cryo-...
Supramolecule | Name: Cytosolic ribosome in the native cellular environment after cryo-FIB lift-out from C. elegans (p1 state) type: complex / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Caenorhabditis elegans (invertebrata) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | tissue |
-Sample preparation
Buffer | pH: 7.3 |
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Vitrification | Cryogen name: NITROGEN Details: The sample was high pressure frozen in a BAL-TEC HPM 100 high-pressure freezer.. |
Details | Adult C. elegans worms. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.5 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 11.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 4895 |
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Extraction | Number tomograms: 5 / Number images used: 13688 / Software: (Name: IMOD, PyTom) |
CTF correction | Software - Name: RELION |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION |